First purification and characterization of a sucrase-isomaltase from goose kidney microvillous membrane.
Goose (Anser anser) kidney microvillus sucrase-isomaltase (EC 3.2.1.48-EC3.2.1.10) was solubilized from isolated microvillus membranes using Emulphogen BC 720 or papain. Detergent-solubilized enzyme (D-SI) was purified 149 +/- 29 times with a yield of 15.7 +/- 2.6% by a two-step procedure which included chromatofocusing. The specific activity was 2.95 +/- 0.34 U/mg protein for sucrase, 1.02 +/- 0.13 for palatinase and 5.63 +/- 0.53 for maltase. D-SI was amphiphilic as indicated by its detergent-binding properties. These properties were not observed for sucrase-isomaltase released from the microvillus membrane by papain. The Mr of the enzyme purified after solubilization by Emulphogen and papain was 543,000 and 380,000, respectively, as determined by gel filtration. The difference in Mr indicates that an Emulphogen micelle is bound to the detergent-solubilized enzyme. In sodium dodecyl sulphate-polyacrylamide gel electrophoresis, sucrase-isomaltase migrated as several polypeptide chains: a major band (Mr 280,000) and at least seven additional minor bands (Mr 220,000-100,000). It is suggested that the major band represents the precursor pro-sucrase-isomaltase and that the lower molecular weight bands are generated by PMSF or aprotinin-resistant proteinases during homogenisation and chromatography of the enzyme. Measured by chromatofocusing, the isoelectric point was found to be pH 4.6. Sucrase-isomaltase accounts for about 20% of total microvillus membrane proteins.
Galand
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Laboratoire de Physiologie Animale, U.F.R. des Sciences Exactes et Naturelles, Reims, France.
D000818:Animals; D002845:Chromatography; D002850:Chromatography, Gel; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D005777:Geese / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007534:Isomaltose / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D007668:Kidney / Q000201:enzymology* / Q000648:ultrastructure; D008320:Maltose / Q000378:metabolism / Q000494:pharmacology; D008871:Microvilli / Q000201:enzymology; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification*; D013393:Sucrase / Q000378:metabolism; D013394:Sucrase-Isomaltase Complex / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D013395:Sucrose / Q000378:metabolism / Q000494:pharmacology
D009097:Multienzyme Complexes; D013395:Sucrose; D007534:Isomaltose; D008320:Maltose; D013394:Sucrase-Isomaltase Complex; D013393:Sucrase; C008189:isomaltulose